A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-O-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-O-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Methylation: a regulator of HIV-1 replication?

Pakistan, the second most populous Muslim nation in the world, has started to finally experience and confront the HIV/AIDS epidemic. The country had been relatively safe from any indigenous HIV cases for around two decades, with most of the infections being attributable to deported HIV positive migrants from the Gulf States. However, the virus finally seems to have found a home-base, as evidenced by the recent HIV outbreaks among the injection drug user community. Extremely high-risk behavior has also been documented among Hijras (sex workers) and long-distance truck drivers. The weak government response coupled with the extremely distressing social demographics of this South-Asian republic also helps to compound the problem. The time is ripe now to prepare in advance, to take the appropriate measures to curtail further spread of the disease. If this opportunity is not utilized right now, little if at all could be done later.     

Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits

While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in cultured mammalian cells. In HEK-293 and COS-7 cells transiently transfected with beta(2)-adrenergic or m2 muscarinic receptors, respectively, treatment with agonist-induced phosphorylation of Akt at serine 473 as evidenced by phosphoserine-specific immunoblots. This effect was blocked by the phosphatidylinositol-3-OH kinase inhibitor LY294002 and wild-type Galpha(i1), and was not duplicated by co-transfection of the constitutively active Galpha(s)-Q227L or Galpha(i)-Q204L mutant. Co-transfection of Gbeta(1), Gbeta(2) but not Gbeta(5) together with Ggamma(2) activated the kinase when assayed in vitro following immunoprecipitation of the epitope-tagged enzyme. In contrast, constitutively activated G protein subunits representing the four Galpha subfamilies were found unable to activate Akt in either cell line. The latter results are in disagreement with a report by Murga et al. (Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem. 273, 19080-19085) that described activation of Akt in response to mutationally activated Galpha(q) and Galpha(i) transfection in COS cells. To the contrary, in our experiments Galpha(q)-Q209L inhibited Akt activation resulting from betagamma or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells Galpha(q)-Q209L transfection inhibited insulin-like growth factor-1 activation of epitope-tagged Akt. In m1 muscarinic receptor transfected HEK-293 cells, carbachol inhibited insulin-like growth factor-1 stimulated phosphorylation at Ser(473) of endogenous Akt in an atropine-reversible fashion. We conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by Gbetagamma heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by Galpha(q). This work identifies Akt as a novel point of convergence between disparate signaling pathways.